Structure-function studies on bovine rhodopsin are to be continued and the position of various functional groups in the sequence determined. The characterization of the acid-soluble cyanogen bromide peptides will be completed. This will include the location of the phosphorylation site(s) and their quantitation. Cysteine residues in rhodopsin will be determined as the C14-N-ethylmaleimide derivatives. A rapid analytical system will be developed which will allow determination of which unique cysteine residues have been modified with any sulfhydryl reagents under any experimental conditions. This will allow clarification of all previous sulfhydryl chemistry of rhodopsin and enable testing of the sulfhydryl-disulfide exchange theory of rhodopsin function. The quantitation and specificity of in vitro phosphorylation of frog rhodopsin will be followed at various extents of bleaching by isoelectric focusing and peptide mapping in order to determine early molecular events. Direct evidence will be obtained for the extent that rhodopsin, rather than only opsin, may be a substrate for the kinase, and how many phosphates are incorporated per molecule of protein. These studies will be extended to in vivo phosphorylation with the same parameters measured plus the reversal of the reaction by phosphatase. The retinal-binding site will be further characterized by isolation and sequencing of N-retinyl-lysine peptides. New techniques for the preparation and characterization of hydrophobic peptides are to be developed and applied to the tryptophan peptides of rhodopsin, some of which are located in the retinal-binding pocket of rhodopsin.